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• Through green synthesis, a new bimetallic licu-ferrite nanospinel was developed. • LiCu-F has a mean particle size of 59.8 nm and a crystallite size of 45.9 nm. • LiCu-F exhibited sufficient antimicrobial activity and can be used in biological applications. • Within 60 min, LiCu-F exhibited bacteriostatic and bactericidal effects on S. aureus and E. coli. • LiCu-F had lower binding energy (−29.16 kcal/mol) for E. coli than for S. aureus (−38.89 kcal/mol). An aqueous carob leaf extract was employed in the current study as a biogenic agent (reducing and capping agents) for the synthesis of novel bimetallic lithium and copper-based nanospinel ferrites (LiCu-F) with antibacterial activity. LiCu-F has a considerable number of surface -OH groups, a BET-specific surface area of 72.5 m2/g, a mean particle size of 59.8 nm, and a crystallinity index of 78.9%, according to the results of the physicochemical characterisation of the ferrite. Within the first 60 min, LiCu-F showed bacteriostatic and bactericidal effects on S. aureus and E. coli based on the time-kill assay. 0.4 mg of LiCu-F demonstrated 67.9% bacteriostatic effects on E. coli while exhibiting 100% inactivation of S. aureus strains, demonstrating that the gram-positive bacteria are more susceptible to LiCu-F. The computational molecular docking analysis demonstrated that LiCu-F has a higher binding affinity for S. aureus (-38.89 kcal/mol) than E. coli (-29.16 kcal/mol), which is consistent with the experimental findings. With an LD 50 of 1000 mg per kg of body weight based on a computational prediction tool, LiCu-F is cytotoxic to bacteria and can cause DNA damage and cell death in bacterial strains. [Display omitted] [ABSTRACT FROM AUTHOR]